The role of RNA polymerase in the regulation of gene expression during growth and sporulation of Bacillus subtilis will be investigated. Since the sigma-43 form of RNA polymerase plays a major role during growth, early stationary phase, and catabolite repression, the following aspects of this enzyme will be investigated in detail: (1) the genetic organization and regulation of the recently cloned and sequenced sigma-43 operon will be analyzed by determining its promoter sites and RNA polymerase specificities, by testing various environmental and physiological conditions that alter transcription from these promoters, by determining the function of the first gene of the operon, and by modifying the promoter regions by site directed mutagenesis; (2) the role of sigma-43 enzyme in catabolite resistant sporulation and catabolite repression will be investigated by studying the relationship between the crsA mutation in the rpoD (sigma-43) gene and the spoO and non-sporulation related suppressor genes for crsA. The studies will involve the isolation of additional suppressor genes for crsA including those which are not related to sporulation. Glucose sensitive promoters will be isolated by a newly developed promoter expression probe plasmid and the effect of the crsA mutation will be tested on the promoters by in vivo and in vitro transcription studies. Those spoO genes which suppress crsA and which can be suppressed by crsA will be cloned and characterized. The effect of spoO gene products will be examined on transcription carried out by the wild type sigma-43 enzyme and the crsA mutated sigma-43 enzyme.
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